9,15-Dioxo-5-cis-prostenoic acid

ABSTRACT

9,15-Dioxo-5-cis-prostenoic acid is formed from 15(S)-hydroxy-9oxo-5-cis-10,13-trans-prostatrienoic acid (PGA2) by the fermentative action of Dactylium dendroides (NRRL 2575). This compound is a diuretic which lacks vasodepressor activity.

United States Patent [191 Hsu et a1.

[ Feb. 25, 1975 9,1S-DIOXO-S-ClS-PROSTENOIC ACID [75] Inventors: CharlesHsu, Skokie; James Jiu;

Seth Setsuo Mizuba, both of Morton Grove, all of 111.

[73] Assignee: G. D. Searle & Co., Chicago, Ill.

[22] Filed: June 25, 1973 [21] Appl. No.: 373,427

Related U.S. Application Data [63] Continuation-impart of Ser. No.295,209, Oct. 5,

1972, Pat. No. 3,788,947.

[52] U.S. Cl. 260/514 D, 195/30, 195/51 R, 260/468 D, 424/317 [51] Int.Cl. C076 61/36 [58] Field of Search 260/514 D, 514 UA, 468 D [56]References Cited UNITED STATES PATENTS 3,773,795 11/1973 Bagli et a1260/3457 OTHER PUBLICATIONS Crubbe et a1., Tet. Letters, 115, 1972.

Primary ExaminerRobert Gerstl Attorney, Agent, or Firm-John J.McDonnell; Elliot N. Schubert [57] ABSTRACT Claim, N0 Drawings 9,l5l1IOXO-5-CIS-PROSTENOIC ACID g-/rat, range 170-210 g. In mostexperiments 2 groups This is a continuation-in-part of our copendingappli- (8 rats) were tested at each dosage level. cation Ser. No.295,209 filed Oct. 5, 1972 now US. The appropriate volume metameter forstatistical Pat. No. 3,788,947. evaluations of response was taken to belog volume. On

9,l5-Dioxo-5-cis-prostenoic acid is formed from 5 the log volume scale,50% reversal of the response to S)-hydroxy-9-oxo-5-cis-10,13-trans-prostatrienoic acid 1 mU ADH/lOO g. was the midpoint between 1mU and (PGA by the fermentative action of the fungus Daccontrol logvolume responses. (This point is equivalent tylium and in particularDactylium dendroides (NRRL* to the geometric mean of the two extremevolume re- 25 75 sponses.) For a test compound, ED was defined as the*NRRL cultures can be obtained at ARS Culture Collection 1815 do enecessar to reduce 507 reversal as defined North University Street,Peoria, Illinois 61604 I l0 y p 0 i NY OH o /on D 1; l1 W PGA SCHEMEIabove, when given together'with 1- mU ADH/lOO g. 9,l5 DiOxO 5 CiSproSten0ic acid is a new and unob The 95% confidence limits of the EDwere calculated vious composition of matter. A 9,15 diOXO prostenoicaccording to Fiellers theorem on the fiducial limits of acid ester isdescribed by J. F. Bagli an'd Tibor Bogri, a ratio Tetrahedron Letters,No. 21 pp. 1639 1969) but in that effect Present Vascular compound thedouble bond is of the trans configura sistance was measured by avariation of the method of tion and located in the 13,14-positon.9,15-Dioxo pros- 25 Brady et Appli h 181645 (1963) Male tanoic acid isalso described in the above reference. It chafles l' rats Welghmg atleast 3 were, fines is recognized in the prostaglandin art that thenumber, themed Wlth urethane gjkg Heparm (l0 sterochemistry and locationof carbon to carbon doumg/kg') as the anncoagulam' B f m' ble bonds inthe side chains dramatically alter the propture was mamtamed at 35 i TheIsolated erties of prostaglandins quarters were autoperfused at aconstant flow rate by PGEZ and PGA2 are examples of prostaglandins meansofaSigmamotor T 8 pump. Systemic and perfuwhich exhibit a diureticeffect which is accompanied Pressures were momtorefl h Statham P23AAwith vasodepression, The prosmglandins, edited by transducers and aSanborn Twm-Viso recorder. With a peter w Ramwen plenum press, New York,1973, constant pump flow, changes in perfusion pressure are Chapter 5.The present compound is distinctive in its h l P PPl' to changes inVascular activity in that it exhibits a diuretic effect without af- Thlssensmve method to measure Changes in Vascufecting vascular resistancei.e. no vasodepression. It reslstahce Caused by Vasopressor andvasodepfessof therefore exhibits selective diuretic activity andselecsubstahcesi i i i a h Sought ft l i the prosta Fermentation isordinarily carried out in the medium i di 40 wherein the organism iscultured. However, it is like- The diuretic properties of the presentcompounds are Wise Possible to Separate the fungal Cells from thedemonstrated by their ability to reverse the action of lure medium ycemrlfugatioh or other means and use idi i hormone (ADH) the resultantcellular matter to implement the fermen- Male Badger rats (BadgerResearch Corp Madison tation. MOIEOVCI', the cells can be rupturedUlt1'21SOl1l- Wi n i w i hi 150-175 w i i d cally or otherwise tofacilitate access to enzymes pres- 70 73F, D i th fi k th a i l wereent, which can be isolated by filtration or extracted ditioned nce tbladd p l ti (b) t i i with a solvent such as acetone or water andsubstituted bation with a French No.8 catheter followed by tap for thega s o Cells thereof. water (5 ml./l00 g.) and (c) 0.5 ml. of 0.9% NaCl,s A nutrient medium is required for culture of the or- On the 7th or 8thday after arrival the first experiment ganism, which is to say onecontaining assimiable nitro- (week I) was performed: 18 hours prior tothe test the g and Carbon; and an q at supply Of Sterile air rats weredeprived of food; but allowed water ad libi- Should be maintainedtherein, for example, by exposing turn. The following day the animals(ca. 180 g./rat, a large surface of the medium to the air or preferablyrange 160-200 g.) were placed in groups of 4 with no passing it throughthe medium in quantities sufficient more than a 2% variation in groupmean weights about to support submerged growth.

the grand mean. At 0 time following bladder palpation Suitable nitrogensources are thus normally emthe animals were (a) loaded orally with0.21% NaCl (5 ployed for the purpose, including soy bean meal,cornml./l00 g.) containing 5% ethanol (v/v) and 5% propysteep liquor,meat extract, protein (optionally dilene glycol (v/v), together withdissolved or suspended gested), peptone, yeast extract, distillerssolubles, catest compound, and (b) 1 milliunit (mU) of Pitressin seinhydrolysate, nitrate, cottonseed meal and/or am- (Parke, Davis & Co.,)per g. in 0.2 ml. of 0.9% monium compounds. All of the foregoingmaterials ex- NaCl. sq. (neck). Sixty minutes later the animals werecepting sometimes the last two serve also as carbon palpated, urinevolume measured, and a second gavage sources. Other carbon-containingsubstances satisfac- (5 ml./l00 g. of 2.5% ethanol in 0.20% NaCl) and aretory and conventionally used as nutrients are the carpeated dose OfADH administered. After 2 hours the 5 boyhydrates, for example,glycerol, glucose fructose, test was terminated by palpation to insurecomplete resucrose, maltose, inositol, dextrin, starch and whey, coveryof a pooled urine sample. Urine volume and Na among which inositol isadditionally useful because of and K excretion were reported in ml./ 100g./2 hours its unusual capacity to stimulate growth.

and uEq/ 100 g./2 hours, respectively. The animals were Phosphate,magnesium, and/or ferrous ions likewise regrouped and retested 1 weeklater (week 11, ca. 200 maybe incorporated in the culture medium asgrowthpromoting factors if desired; buffers may be added to assure thatgrowth is initiated at a suitable pH; and wetting agents may be employedto improve contact between the prostaglandin and the fermenting agent.An anti-foaming agent is usually beneficial. Where isolated cells orenzymes are used to induce fermentation rather than the intact andgrowing organism, nutrients need not, of course,'be present; but ineither solvent the medium is customarily preponderantly aqueous.

A preferred embodiment of the present invention is conducted in a mediumconsisting of 150 parts of cottonseed meal, 65 parts by volume ofcornsteep liquor, 50 parts of Dextrose, 0.3 parts by volume of 6Nhydrochloric acid, and 1000 parts by volume of water is sterilized byheating for 10 min. at 121, whereupon it is cooled to 23 i 1 and then isinoculated with 10 parts of a fluid culture of Dactylium dendroides(NRRL 2575). The inoculating fluid is prepared by incubating a seedculture for 72 hours in 100 parts by volume of the above mentionedsterilized medium from spores originating on an agar slant.

The inoculated medium is incubated for 1.68 hours and then 0.1 parts ofl(S)-hydroxy-9-oxo-5-cis-10,13-

trans-prostatrienoic acid (PGA is added. Incubation in the presence ofthe PGA substrate is continued for 24 hours, at which time the mixtureis extracted with dichloromethane. The dichloromethane layer isseparated and the solvent is removed from this separated layer byevaporation in vacuo.

When the fungus, Dactylium dendroides (NRRL 2575), is incubated in theabove medium for 48 hours prior to the addition of 0.1 parts ofl5(S)-hydroxy-9- oxo-5-cis-10,13-trans-prostatrienoic acid (PGA theproducts are 9,15-dioxo-5 cis-prostenoic acid and 9,1-5-dioxo-5-cis-8(12)-prostadienoic acid. In both fermentations theproducts are isolated by silica gel chromatography.

The following examples are presented to further illustrate the presentinvention. They should not be con strued as limiting it either in scopeor in spirit. In these examples quantities are indicated in parts byweight unless parts by volume are specified, and temperatures areindicated in degrees Centigrade (C.).

EXAMPLE 1 A medium consisting of 150 parts of cottonseed meal, 65 partsby volume of cornsteep liquor, 50 parts of Dextrose, 0.3 parts by volumeof 6N hydrochloric acid, and 1000 parts by volume of water is sterilizedby heating for 1 hour at 121, whereupon it is cooled to 23 1 and then isincubated with 10 parts of a fluid culture of Dactylium dendroides (NRRL2575 The inoculating fluid is prepared by incubating a seed culture for72 hours in 100 parts by volume of the above mentioned sterilized mediumfrom spores and mycelium originating on an agar slant.

The inoculated medium is incubated for 168 hours and then 0.1 parts of15(S)-hydroxy-9-oxo-5-cis-I0, l 3- trans-prostatrienoic acid (PGA isadded. Incubation in the presence of the PGA substrate is continued for24 hours, at which time the mixture is extracted with dichloromethane.The dichloromethane layer is separated and the solvent is removed fromthis separated layer by evaporation in vacuo. The crude extract is takenup in 850 parts by volume of phosphate buffer solution of pH 8 and thissolution is extracted with hexane. The aqueous layer is made acidic with6N hydrochloric acid to pH 2.5 and extracted with dichloromethane. Thedichloromethane is removed by evaporation in vacuo. The remainingmaterial is taken up in ethyl acetate and that which is insoluble inethyl acetate is removed by filtration. The ethyl acetate is evaporatedand the remaining material is taken up in a solution consisting of 97parts by volume of benzene. 2 parts by volume of dioxane, and 1 part byvolume of acetic acid. This solution is placed on a chromatographiccolumn packed with silica gel and 9,l5-dioxo- 5-cis-prostenoic acid iseluted in a solvent system consisting of 92.3 parts by volume ofbenzene, 6.3 parts by volume of dioxane, and 1.4 parts by volume ofacetic acid. 9,l5-Dioxo-5-cis-8(12)-prostadienoic acid is eluted in asolvent system consisting of 87 parts by volume of benzene, 10 parts byvolume of dioxane, and 3 parts by volume of acetic acid.

EXAMPLE 2 The procedure set out in Example 1 is followed with theexception that the Daclylium dendroides (N RRL 2575) is allowed to growonly for 48 hours before addition of 0.1 parts ofl5(S)-hydroxy-9-oxo-5-cis-l0,l3- trans-prostenoic acid. The fermentationmixture is extracted and is prepared for column chromatography on silicagel also as described in Example 1. 9,15-Dioxo-5- cis-prostenoic acid iseluted in a solvent system consisting of91 parts by volume of benzene, 7parts by volume of dioxane, and 2 parts by volume of acetic acid. 15(S)-Hydroxy-9-oxo-5-cis-l3-trans-prostadienoic acid is eluted in a solventsystem consisting of parts by volume of benzene, 10 parts by volume ofdioxane, and 5 parts by volume of acetic acid.

What is claimed is:

1. 9,15-Dioxo-5-cis-prostenoic acid.

1. 9,15-DIOXO-5-CIS-PROSTENOIC ACID.